Cellular surveillance for the integrity of genetic information passed from parental cells to subsequent generations is carried out by a network of proteins primarily involved in cell-cycle regulation, DNA replication, DNA repair, and chromosome segregation. Defects of any protein factor in this network render cells to genetic instability to different extents, and therefore, predispose to cancer. One of the involved pathways is the DNA mismatch repair (MMR) system that has a crucial role in maintaining genome stability by monitoring and correcting mismatched nucleotides occurred during DNA recombination and DNA synthesis. Mutations in MMR genes can cause higher mutation rates and increase microsatellite instability. Germline mutations in several MMR genes, including MutS and MutL homologues, contribute to the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC) in humans, with mutations in hMLH1 and hMSH2 genes representing major contributors to HNPCC. Our recent work has revealed a direct physical link between hMLH1 and hMRE11 both in vitro and in vivo. Human hMRE11 represents an essential nuclease of the multifunctional protein complex (hRAD50-hMRE11-NBS1) that promotes repair of DNA double-strand breaks and plays a role in the signaling of DNA damage response as well as in meiotic recombination and telomere maintenance. Mutations in hMRE11 contribute to the development of the rare "AT-like" (ATLD) disorder. It is known that hMRE11-associated complex displays dynamic spatial and temporal cellular distribution and form DNA-damage induced foci at the sites of DNA damage. Therefore, the long-term goal of this research program is to elucidate the molecular mechanisms involved in MMR and the functional links between MMR proteins and the hMRE11-associated protein complex, as well as the biological effects of mutations that disrupt the interplay among these protein factors. We propose to address these important issues through a vigorous systematic approach that includes the following specific aims: (1) to test the hypothesis that hMRE11 represents a functional component of the DNA mismatch repair pathway; (2) to determine whether hMLH1 binding stimulates nuclease activities of hMRE11 and the functional roles of hMRE11 nuclease activities in the process of MMR; (3) to test the hypothesis that hMLH1 HNPCC missense mutations differentially affect the formation of hMLHI-hMRE11 and hMLHI-hPMS2 heterocomplexes, and therefore disruption of hMLH1-hMRE11 complex represents an alternative mechanism underlying the pathogenic effects of hMLH1 HNPCC mutations. The results of the proposed studies will provide insight into the molecular mechanisms by which MMR and the hMREll-associated complex act together to maintain genetic stability, as well as to provide a better understanding of hMLH1 and/or hMRE11 deficiencies in human diseases such as cancer.